Optimization of Transformation of soybean somatic embryos via particle bombardment
1998
Sontichai Chanprame(Kasetsart University. Kamphaeng Saen Campus, Nakhon Pathom (Thailand). Faculty of Agriculture. Department of Agronomy) | Widholm, Jack M.(University of Illinois at Urbana-Champaign, IL (USA). Department of Crop Sciences)
Somatic embryos of soybean (Glycine max L.) Jack were bombared with 1.0 micro m diameter tungsten particles coated with the plasmid pZA-300 encoding beta-glucuronidase (GUS) and hygromycin phosphotransferase (HPT), 10 and 20 days after bombardment the GUS histochemical assay was performed, in the DNA precipitation procedure, 2.5 M CaCl2, pH 5, resulted in higher levels of transient GUS-expression than 2.5 M CaCl2, pH 10 or 2.5 M Ca(NO3)2, pH 5 or 10. Helium gas pressures between 900 and 1,300 psi did not produce significant difference in GUS-expression, but the lowest presure produced less tissue damage. However, 20 days after bombardment, the 1,100 psi treatment gave the highest number of blue spots (GUS positive) and produced a higher level of stable transformation as well. When 1,100 psi was used, the distance between the rupture disk and the target, and between the rupture disk and the macrocarrier, were 13 cm and 8 mm, respectively. The flying distance (distance between the macrocarrier and the stopping screen) was 6 mm. The use of these parameters has resulted in the efficient production of transgenic embryogenic suspension cultures.
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Эту запись предоставил Kasetsart University